There are basically two different types of heart valve prostheses: Prostheses comprising mechanical valves, which are artificially produced, usually being made of graphite coated with pyrolytic carbon, and prostheses comprising valves made of biological tissue, usually pericardial tissue, which is usually obtained from animal sources (e.g. swine or cattle). The heart valve formed of biological tissue is usually mounted in a base body (e.g. a rigid plastic framework or a self-expanding stent), which is then implanted at the position of the natural valve. The present invention describes a method for preparing such tissue for use in a heart valve prosthesis for implantation at the site of a natural heart valve.
The tissue of origin must be thoroughly cleaned and prepared before implantation. In so doing, the tissue is modified, to the greatest extent possible, such that the tissue is not recognized by the body as foreign tissue, is not calcified, and has the longest life span possible. Such a method for preparing tissue substantially comprises at least two main steps having a plurality of intermediate rinsing processes.
The first essential preparation step is the so-called decellularization of the tissue. In this step, cell membranes, intracellular proteins, cell nuclei, and other cellular components are removed as completely as possible from the tissue in order to obtain the purest extracellular matrix possible. Any cells and cellular components remaining in the tissue would be potent crystal nuclei, in particular, for an unwanted calcification of the biological implant material. The decellularization, as a washing step, should be performed in a manner that is so gentle that the structure and the collagen fibers in the extracellular matrix remain as unaffected as possible while ensuring that all cells contained therein are thoroughly removed from the tissue.
The second essential preparation step is that of cross-linking the tissue, in particular the collagen fibers. After decellularization, preferably all cellular components have been removed from the tissue and the biological material nearly exclusively comprises the extracellular matrix. In the case of pericardial tissue, the extracellular matrix is formed primarily of collagen fibers. In order to obtain biological material having the most optimal mechanical properties possible and to prevent rejection reactions by the receiving body, the collagen fibers are cross-linked by means of a suitable cross-linking agent via the incorporation of chemical bonds. The cross-linking agent binds to the amino groups of the collagen fibers and forms chemically stable compounds between collagen fibers. A biological material having long-term stability is thereby obtained from the three-dimensionally arranged collagen fibers, wherein this biological material is no longer recognized as foreign biological material. The stability and strainability of the tissue is markedly increased by means of the three-dimensional cross-linking or linking of the individual collagen fibers via the cross-linking agent. This is decisive, in particular, in the case of use as tissue of a heart valve, where the tissue must open and close, in brief intervals, as a valve.
An alternative method for preparing biological tissue is described in WO 2004/052417. In this method according to the prior art, the tissue is decellularized with a 1-2% deoxycholic acid solution. After a plurality of rinsing steps, the tissue is conditioned in a solution containing a cyclic lipopeptide. The cyclic lipopeptide surfactin is used, in particular, as the conditioning agent after decellularization is completed and before repopulation with cells. In this alternative method according to the prior art, cross-linking of the collagen fibers is not performed using a suitable cross-linking agent. After conditioning, the tissue is populated with natural cells.
WO 2011/109433 discloses a method for preparing biological tissue, wherein distilled water is used for decellularization. Glutaraldehyde functions as the cross-linking agent in a subsequent step.
WO 2005/118014 discloses the use of a first ionic detergent and a second non-ionic detergent for decellularization. In this case, an anionic detergent such as sodium dodecyl sulfate or sodium dodecyl sulfonate is preferably provided as the first ionic detergent. As an alternative, bile acids such as sodium cholate or sodium deoxycholate can be used as the first detergent. The second detergent is electrically neutrally charged, such as a detergent containing polyethylene glycol.
While strong detergents may thoroughly decellularize tissue, they also tend to weaken the mechanical properties of the tissue itself. However, using more gentle detergents risks incomplete decellularization or incomplete removal of cellular components, thereby leaving antigenic moieties on the tissue. We have found that the above-mentioned calcifications can still occur even in spite of carefully performed decellularization procedures and can originate, inter alia, in antibodies directed against galactose-α-1,3-galactose-β-1,4-N-acetylglucosamine epitopes (α-gal epitopes on the surface of the implanted tissue). Here, α-gal epitopes can lead to severe immune responses that encourage calcification. The concentration of α-gal epitopes on the surface could be reduced in principle by harsh decellularization conditions, but this would have a significantly negative influence on the mechanical properties of the valve material. In order to minimize calcifications and provide tissue having significantly improved mechanical properties, it would therefore be desirable to provide tissue that has been subjected to gentle decellularization and with which α-gal epitopes on the surface of the tissue have been fully removed where possible.
Accordingly, there remains a need for improved methods for the treatment of biological tissue to prevent or reduce calcification while improving or maintaining the mechanical stability of tissue for implantation.